1. Field of the Invention
The present invention relates to a process for producing an antithrombin III composition comprising an antithrombin III molecule having complex type N-glycoside-linked sugar chains, wherein the complex type N-glycoside-linked sugar chains have a structure in which fucose is not bound to N-acetylglucosamine in the reducing end in the sugar chains.
2. Brief Description of the Background Art
Thrombus formation accompanies a danger of stopping blood flow. Since cutoff of blood flow by the formation of thrombi becomes a lethal factor, the living body has several mechanisms to control and regulate blood coagulation. That is, direct inactivation of activated coagulation factors by serine protease [The Thrombin, Volume I (Machovich R., ed.), pp. 1-21, CRC Press, Boca Raton (1982)], a regulatory mechanism based on the degradation of factor V and factor VIII by activated protein C [Progress in Hemostais and Thrombosis, Volume 7 (Spaet T. H., ed.), pp. 25-54, Grune & Stratton, New York (1984)] and an inhibitory mechanism of activated coagulation factors by various serine protease inhibitors in blood. In addition, the presence of a tissue factor inhibitor which inhibits activation of factor VII in an activated factor X-dependent manner [Journal of Japanese Society on Thrombosis and Hemostasis, 2, 550 (1991)] has also been found. The most important mechanism among these is the inhibitory mechanism of activated coagulation factors by various serine protease inhibitors in blood.
Various serine protease inhibitors are present in blood, and their amount reaches 10% of the total plasma protein. It is known that 4 inhibitors among these inhibitors, namely antithrombin III, α1 proteinase inhibitor, α2 macroglobulin and heparin cofactor II, are important in regulating blood coagulation. Among such inhibitors, antithrombin III is particularly important and occupies 70% of the antithrombin activity in plasma.
Antithrombin III is a glycoprotein comprising 432 amino acids and having a molecular weight of approximately 59,000 to 65,000, and has three disulfide bonds, Cys8-Cys128, Cys21-Cys95 and Cys247-Cys430, in its molecule [Proc. Natl. Acad. Sci., USA, 80, 1845 (1983)). By these bonds, a large loop structure is formed on the C-terminal, and an Arg393-Ser394 bond is present as the active center in this loop structure (FIG. 1). Human antithrombin III has an isoelectric point of 5.11. N-Glycoside-linked sugar chains are added to 4 positions, the 96th, 135th, 155th and 192nd asparagine residues counting from the N-terminus (hereinafter referred to as Asn96, Asn135, Asn155 and Asn192, respectively) of antithrombin III. The antithrombin III in human plasma exists in two kinds of isoforms, an α type having four N-glycoside-linked sugar chains and a β type having only three N-glycoside-linked sugar chains but not having a sugar chain to the Asn135 [Pathophysiol. Haemost. Thromb., 32, 143 (2002)), and in the antithrombin III in human plasma, 90 to 95% is the α type and the remaining 5 to 10% is the β type.
The complex type N-glycoside-linked sugar chains added to antithrombin III are constituted by N-acetylglucosamine, sialic acid, galactose and mannose (FIG. 2). One of the characteristics of the antithrombin III distributing in human plasma is that its sugar chain structure is free from the fucose modification.
Antithrombin III has been developed as a blood coagulation inhibitor and is broadly used in the world for the treatment of thrombosis based on congenital antithrombin III deficiency and multiple intravascular blood coagulation syndrome which accompanies reduction of antithrombin III.
Blood preparations such as antithrombin III are produced by using pooled human plasma samples as the raw material. In Japan, the pooled plasma is prepared at Plasma Fractionation Center, Japanese Red Cross Society, by mixing plasma samples of approximately 5,000 to 10,000 volunteers after completion of the 6 months of storage, and provided. In reality, in order to produce one lot of a blood preparation such as a dry concentrated human blood coagulation factor VIII preparation, Cross Eight M (Japanese Red Cross Society), several batches of cryoprecipitates obtained from the above-described pooled plasmas are necessary, and plasma samples of approximately 80,000 volunteers are used [Japanese Journal of Transfusion Medicine, 48, 27 (2002)].
The pooled plasma is produced by using blood samples provided by blood donors as the raw material, and it has been reported that the human parvovirus B19-positive ratio in blood donors in Japan is estimated to be 0.6 to 0.8% [Journal of Japan Society of Blood Transfusion, 42, 231 (1996)]. Thus, it is calculated that one lot equivalent to the above-described Cross Eight M is contaminated with human parvovirus B19-positive blood samples corresponding to roughly 480 to 640 donors. The human parvovirus B19 is a small virus of 18 to 26 nm in diameter without envelope, and keeps its resistance even after carrying out heat treatment at 60° C. for 30 minutes, acid treatment at approximately pH 3, chloroform treatment, surfactant treatment and the like [Science, 262, 114 (1993)], so that it cannot be eliminated by general virus elimination methods. Accordingly, elimination of human parvovirus B19 requires a step for filtration through an exclusively developed virus eliminating membrane having a pore size of several nanometers to several ten nanometers. However, it is considered that a filtration step which uses such a small membrane pore size, namely a nano-filtration step, is difficult to be introduced into the production process of many plasma fractionation preparations [Japanese Journal of Transfusion Medicine, 48, 27 (2002)]. It is considered that human parvovirus B19 is the cause of erythema infectiosum, and generally shows only transient cold-like symptoms in the case of healthy persons without anti-B19 antibody, but causes chronic hemolytic anemia in some cases. Also, it is said that it sometimes induces serious acute pure red cell aplasia in immunodeficiency patients. In addition, there is a report stating that pregnant women having no anti-B19 antibody sometimes result in miscarriage or the unborn babies cause edema, and 15% of the intrauterine fetal death was positive regarding the result of DNA inspection of B19 [Lancet, 357, 1494 (2001)]. In the dry concentrated human blood coagulation factor VIII preparation, Cross Eight M (Japanese Red Cross Society), a case in which a transient infection with human parvovirus B19 by the administration of this preparation was suspected was reported in September, 1997 [Journal of Japanese Society of Child Hematology, 11, 289 (1997)].
A hepatitis B virus-negative, hepatitis C virus-negative and human immunodeficiency virus I and II-negative pooled plasma is used as the production material of antithrombin III blood preparations such as Neuart (manufactured by Mitsubishi Pharma Corporation) and Anthrobin P (manufactured by Aventis Boehring), but the presence or absence of human parvovirus B19 in the raw material has not been confirmed.
Although a virus inactivation treatment at 60° C. for 10 hours, namely pasteurization, is carried out in the production process of antithrombin III blood preparations, there are problems such that the antithrombin III which is a protein is denatured, and AIDS virus, human parvovirus and prion which becomes the cause of mutation type Creutzfeldt-Jacob disease cannot be completely removed.
As described in the above, the use of blood preparations has disadvantages in that there is a risk of viral infection, and the risk cannot completely be excluded by the current techniques. Thus, an antithrombin III preparation with improved safety is in demand.
Accordingly, in order to provide human antithrombin III without using human plasma as the raw material, its replacement with a recombinant has been considered. However, the activity of the recombinant antithrombin III prepared by using gene recombination techniques is inferior to the activity of antithrombin III obtained from a natural material such as plasma. This is because it is considered that the sugar chain structure to be added to the recombinant is different from that of antithrombin III prepared from plasma, and specifically, it is assumed that since fucose is bound to the complex type N-glycoside-linked sugar chains to be added to the recombinant antithrombin III, its affinity with heparin becomes low, and therefore sufficient anti-blood coagulation activity cannot be obtained [Journal of Biological Chemistry, 268, 17588 (1993), Biochemistry, 35, 8881 (1996)). To date, there are reports on recombinant antithrombin III produced by a baby hamster kidney-derived BHK cell [Journal of Biological Chemistry, 268, 17588 (1993), Biochemistry, 35 8881 (1996)], a Chinese hamster ovary-derived CHO cell (WO02/02793) or a transgenic goat (US 2003096974), but fucose is bound to N-acetylglucosamine in the reducing end in the complex type N-glycoside-linked sugar chains binding to the recombinant antithrombin III in each of these cases. The ratio of the complex type N-glycoside-linked sugar chains to which fucose is bound to the total complex type N-glycoside-linked sugar chains in the produced recombinant antithrombin III varies depending on the host cell, but is estimated to be from 39 to 95%. Attempts have been made to reduce the ratio of the sugar chain in which fucose is bound to the complex type N-glycoside-linked sugar chains by various devices such as improvement of the culturing method, but it has not been succeeded yet in producing a recombinant antithrombin III having a sugar chain structure equivalent to that of the natural antithrombin III.